Research in Progress Currently, there are three main ongoing projects in the lab: The first project is focused on elucidating mechanistic details of the interaction between type II topoisomerases and DNA. One aspect of this interaction concerns the ability of type II topoisomerases to relax the topology of DNA to below equilibrium values. In vivo these topoisomerases are responsible for unlinking replicated chromosomes prior to cell division. Since even a single link between sister chromosomes can prevent division and induce cell death, it is important that these enzymes preferentially unlink rather than link DNA molecules. In vitro it was shown that this was the case, but the mechanism remains a mystery. One proposed mechanism for this topology simplification posits that the topoisomerase induces a sharp bend in the DNA on binding, which would favor unlinking over linking. Using atomic force microscopy (AFM), we have measured the DNA bend angle imposed by the binding of three type II topoisomerases from different organisms (Human, Yeast, and E. coli). The measured bend angles do not support the bend angle model of topology simplification. This is an important finding as the experimental evidence to date has been equivocal concerning the bend angle model. We have also developed new quantitative analysis methodologies for AFM images of protein DNA complexes. These include an image-processing based analysis of the DNA bend angle from AFM images that is faster and less prone to artifact than the currently used manual methods. Currently, we are testing two alternative models of topology simplification. The models postulate either a kinetic proofreading mechanism in which the topoisomerase catalyzes strand passage only after repeatedly encountering a DNA segment, or a mechanism in which the topoisomerase specifically recognizes DNA in a hooked juxtaposition geometry. To test these models we are using magnetic tweezers to measure the unlinking of two DNA strands wrapped around each other a specific number of times under a controlled force. By measuring the rate of strand passage as a function of the imposed geometry and force and performing Monte-Carlo simulations to obtain the distribution of DNA configurations for each condition, we can test both models. We anticipate that these experiments will unambiguously confirm or refute the two competing models for non-equilibrium topology simplification by type II topoisomerases. A second aspect of the interaction between type II topoisomerases and their DNA substrates concerns the diverse topological activities exhibited by type II topoisomerases that share a common mechanism. These activities include the symmetric relaxation of positively and negatively supercoiled DNA by most type II topoisomerases, the introduction of negative supercoils by DNA gyrase, and the asymmetric relaxation of negative and positive supercoils by some type II enzymes. These differences in activity are believed to arise from differences in the C-terminal domains (CTDs), but the molecular basis underling these variations in activity have not been elucidated. We have produced a series of CTD mutants of E coli Topoisomerase IV (Topo IV). We are employing a combination of ensemble and single molecule assays to test the effects of these mutations on the substrate selectivity. In collaboration with Neil Osheroff at Vanderbilt University, we are also investigating the mechanism of chiral sensing by human type II topoisomerase (hTopo II). By measuring the relaxation of individual DNA molecules by hTopo II with magnetic tweezers, we determined that the mechanism of chiral discrimination by this enzyme is due to a salt-dependent difference in relaxation rates between positively and negatively supercoiled DNA. This was surprising given that chiral discrimination by E. coli Topo IV results from differences in processivity rather than relaxation rate. The second project is focused the mechanisms underlying multi-enzyme complex activity. RecQ helicases and topoisomerase III have been shown to functionally and physically interact in organisms ranging from bacteria to humans. Disruption of this interaction leads to severe chromosome instability, however the specific activity of the enzyme complex is unclear. Analysis of the complex is complicated by the fact that both the helicase and the topoisomerase individually modify DNA. The ability of single-molecule techniques to measure the activity of a single enzyme or enzyme complex in real time is well suited to the study of such complicated processes in which multiple activities may occur over multiple time scales. We are using single-molecule fluorescence techniques to measure the unwinding kinetics and step size of RecQ helicase alone and in the presence of Topo III. These experiments and the will pave the way for experiments in which the activity and the association state of single enzymes and complexes will be assayed simultaneously using a combination of single molecule manipulation and single molecule visualization techniques. In the third project, a collaboration with Gregory Goldberg at Washington University St. Louis, we employed single-molecule TIRF to study the motion of single matrix metalloproteinases (MMPs) during the digestion of collagen. MMPs play an important role in physiological collagen processing pathways including tissue remodeling, wound healing and cell migration. However, the mechanistic details of MMP interactions with collagen have been refractory to study due to the complex nature of the collagen substrate and the motion of the MMPs. By tracking individual MMPs on isolated collagen fibers with high spatial and temporal resolution we could characterize the motion of the MMP on the substrate, and how this motion is coupled to proteolytic activity. This approach has provided detailed mechanistic information for this important class of enzymes. We have for the first time observed the complex motion of individual MMPs on collagen fibers and have developed a comprehensive quantitative model describing how this motion is coupled to proteolysis of the collagen fiber. We found that the motion of MMPs on collagen is both biased and hindered diffusion, that there are binding hot-spots for MMPs on collagen spaced 1 micron apart, and that the motion of MMPs on collagen is interrupted by long pauses of duration 1 second. These results were unanticipated and provide unprecedented insight into the interaction of MMPs with collagen while highlighting the unique capabilities of single-molecule methods to measure complex biomolecular processes. The initial measurements and comprehensive modeling are complete and we are writing the first manuscript. Furthermore, we developed new methodologies to treat diffusion in single-molecule traces, which are applicable to any single-molecule analysis of diffusion trajectories. Future work on the MMP tracking will be focused on improving the temporal and spatial resolution of the tracking in addition to extending the duration of individual trajectories through the use of quantum dot labels, or nitrogen vacancy nano-diamond labels. Future research goals: Our immediate goal is the completion of the ongoing projects in the lab. Longer term goals include the development of a new optical trap and magnetic tweezers instrument combined with single-molecule fluorescence detection capabilities. This instrument will permit simultaneous measurements of the activity and composition of multi-enzyme complexes interacting with a single DNA molecule. This instrument will open up other areas of research including the possibility of observing the dynamics of supercoiled DNA. As a first step in this direction, we are in the process of combining a magnetic tweezers instrument with single-molecule fluorescence detection capabilities.